Regulation of the length of the fertile period in the domestic fowl by numbers of oviducal spermatozoa, as reflected by those trapped in laid eggs

The numbers of spermatozoa trapped in the vitelline membrane of laid eggs were counted after staining with the fluorochrome 2,4-diamidino-2-phenylindole. In a group of 24 hens inseminated with different numbers of spermatozoa to produce different lengths of fertile periods, the numbers of spermatozoa in successive eggs from each hen decreased logarithmically with respect to days following insemination. A relationship could be described between the numbers of spermatozoa per unit area of membrane of an egg and the probability of that egg being fertile. After insemination the number of spermatozoa on successively-laid eggs appears to become reduced until a critical value is reached, after which the hen will lay infertile eggs. By estimating the day on which the critical value was achieved, the actual length of the fertile period could be predicted. It is suggested that the numbers of spermatozoa trapped in the vitelline membrane of laid eggs represent those which surround the ovum at the time of fertilization.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Metabolism of high density lipoproteins by the perfused rabbit liver

The role of the liver in the catabolism of high density lipoproteins (HDL) was examined in isolated perfused rabbit livers. Using 125I-labeled rabbit HDL the disappearance of labeled apolipoproteins from the perfusate was biphasic with 7% of the label removed after 20 min and a further 6% between 20 and 90 min. In contrast, with HDL labeled with [3H]cholesteryl esters 35% of label had been removed after 90 min. The effect of liver perfusion on HDL size and composition was further studied by recirculating rabbit HDL for 120 min. In control experiments HDL was incubated at 37 degrees C for 120 min with nonperfused media and with media that had been liver perfused. The added HDL was predominantly particles of 4.8-4.9-mm radius, and incubation with nonperfused and preperfused media produced no significant change in size. However, liver perfusion resulted in particles predominantly 4.2-4.3-mm radius. Hepatic perfusion also significantly reduced HDL cholesteryl ester composition as a percentage of lipoproteins mass from 13.3 +/- 2.2% in control incubations to 10.7 +/- 3.1% (p less than 0.001), and cholesteryl ester:protein mass ratio was reduced from 0.31 +/- 0.06 in control to 0.24 +/- 0.10 (p less than 0.001) after 120 min of liver perfusion. Thus interaction of rabbit HDL with rabbit liver results in smaller HDL particles significantly depleted of core cholesteryl esters.     

A simple method to quantitatively measure polypeptide JHNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J coupling constant and the linewidth (_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J = 0.5(_1/2) – MW/5000 + 1.8 for TOCSY spectra and J = 0.6(_1/2) – MW/5000 – 0.9 for NOESY spectra, where _1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which _1/2 measurements can be made, means that J coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date.     

The effect of premature and delayed birth on the development of UDP-glucuronosyltransferase activities towards bilirubin, morphine and testosterone in the rat.

In the rat, UDP-glucuronosyltransferase activities towards bilirubin, morphine and testosterone increase markedly after normal or premature birth. This rapid development is superimposed upon a much slower maturation of activity which occurs in utero during the last 2 days of normal gestation and gestation when birth is delayed. Development of all three activities is similar under these different conditions, suggesting a common developmenpal regulatory mechanism.     

Regulation of onset of development of UDP-glucuronosyltransferase activity towards o-aminophenol by glucocorticoids in late-foetal rat liver in utero.

1. A precocious development of UDP-glucuronosyltransferase activity (EC 2.4.1.17) towards o-aminophenol is demonstrated in 15-17 day foetal rat liver in utero after dexamethasone administration to the mother. 2. This stimulation of liver transferase activity in utero is directly proportional to the dose of dexamethasone infected. 3. Precocious development of transferase activity in utero can also be effected with the natural glucocorticoid cortisol by multiple injections of large amounts of this hormone into the mother. 4. Transferase activity towards o-aminophenolin foetal lung, kidney and upper alimentary tract can also be precociously stimulated by dexamethasone in 17-day foetuses in utero. 5. Natural development of hepatic transferase activity between days 18 and 20 of gestation is retarded after foetal hypophysectomy by decapitation in utero. 6. Overall glucuronidation of o-aminophenol, as observed in foetal rat liver, is also precociously stimulated by dexamethasone. 7. From this and from evidence previously presented we suggest that glucocorticoids, which are known to increase in rat foetuses between days 17 and 20 of gestation, trigger the normal development in utero of hepatic transferase activity towards o-aminophenol which occurs at that time. We also suggest that these hormones are responsible for the rise in activity of the enzyme in foetal lung, kidney and upper alimentary tract which occurs during the same gestational period.     

Functional heterogeneity of UDP-glucuronosyltransferase as indicated by its differential development and inducibility by glucocorticoids. Demonstration of two groups within the enzyme''s activity towards twelve substrates.

1. UDP-glucuronosyltransferase activity towards 12 substrates has been assessed in rat liver during the perinatal period. 2. Between days 16 and 20 of gestation, enzyme activities towards the substrates 2-aminophenol, 2-aminobenzoate, 4-nitrophenol, 1-naphthol, 4-methylumbelliferone and 5-hydroxytryptamine (the 'late foetal' group) surge to reach adult values, while activities towards bilirubin, testosterone, beta-oestradiol, morphine, phenolphthalein, and chloramphenicol (the 'neonatal' group) remain negligible or at less than 10% of adult values. 3. By the second postnatal day, enzyme activities towards the neonatal group have attained, or approached adult values. 4. Dexamethasone precociously stimulates in 17-day foetal liver in utero transferase activities in the late foetal, but not the neonatal group. A similar inductive pattern is found for 15-day foetal liver in organ culture. 5. It is suggested that foetal glucocorticoids, whose synthesis markedly increases between days 16 and 20 of gestation, are responsibile for triggering the simultaneous surge of all the hepatic UDP-glucuronosyltransferase activities in the late foetal group. The neonatal group of activities apparently require a different or additional stimulus for their appearance. 6. The relationship of these two groups of transferase activities to other similar groups observed during induction by xenobiotics and enzyme purification is discussed.