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Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [(3)H]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [(3)H]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [(3)H]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide. Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel.

A panel of twenty human testis cDNA clones were expressed in an Escherichia coli expression system and six clones were found to express identifiable fusion polypeptides. Expression was found to be influenced not only by the site of localization of the polypeptide in the host cells, but also by the temperature used for induction. This emphasized the need for cytoplasmic and periplasmic expression of new antigens of unknown properties, as well as the use of temperatures of 30°C or lower. A majority of the expressed polypeptides were mainly in an insoluble form. By reducing the induction temperature to 30°C production of the soluble fraction was further improved.

ABSTRACT: The possibility of contaminating antibodies in an antiserum causing reactions in countercurrent immunoelectrophoresis at low antigen concentrations, though not at higher concentrations, was raised, and also argued against, in a recent, widely-publicized legal case.This study has demonstrated that, in this technique, precipitin bands can be produced in tests involving low concentrations of antibodies only where there are also low concentrations of antigen, not where there are higher concentrations of antigen. These results indicate that, under appropriate circumstances, in counter-current Immunoelectrophoresis, a low concentration of contaminating antibodies in an antiserum could produce a precipitin band with a low concentration of an unsuspected antigen. This possibility points up the necessity of establishing the identities of the reacting antigens and antibodies in such tests, using a different procedure, such as the double radial immunodiffusion (Ouchterlony) technique. It is suggested that the value of counter-current immunoelectrophoresis in forensic testing should be given consideration.

A comparison is made between some properties of immunoglobulin and nonimmunoglobulin sperm-agglutinins. It is found that antisperm antibodies are directed against spermatozoal components and bind equally well to viable and nonviable spermatozoa. On the other hand nonimmunoglobulin sperm-agglutinins are directed at both spermatozoal and seminal plasma components and bind to viable sperm. β-lipoprotein may not be involved in the nonimmunoglobulin sperm-agglutinin found in human sera.

Regulation of Escherichia coli carbamyl phosphate synthetase by UMP and IMP was examined in studies with various analogs of these nucleotides. Whereas UMP inhibits enzyme activity, the arabinose analog of UMP was found to be an activator. dUMP neither activates nor inhibits, but binds to the enzyme in a manner similar to UMP as evaluated by direct binding studies, sedimentation behavior, and ultraviolet difference spectral measurements. dUMP decreases inhibition by UMP and activation by IMP, but has no effect on activation by L-ornithine. The findings are in accord with the view that IMP and UMP bind to the same region of the enzyme; a possible general model for such overlapping binding sites is considered. Additional evidence is presented that inorganic phosphate can modulate regulation of the activity by nucleotides. Phosphate (and arsenate) markedly increase inhibition by UMP, decrease activation by IMP, but do not affect activation by L-ornithine. The extent of activation by IMP and by L-ornithine and that of inhibition by UMP are decreased when Mg2+ concentrations are increased relative to a fixed concentration of ATP. The findings suggest that the allosteric effectors may affect affinity of the enzyme for divalent metal ions as well as, as previously shown, the affinity of the enzyme for Mg-ATP.

64Cu(II) is extensively absorbed and excreted, mainly in the faeces, when applied to the skin of rats as complexes with (a) salicylate in ethanol with dimethylsulphoxide and glycerol or (b) phenylbutazone in dimethyl sulphoxide and glycerol, previously found to be anti-inflammatory.64Cu distribution paralleled that observed after administering aqueous64Cu-salicylate i.m. It is concluded that copper(II) can pass rapidly through the dermal barriers when applied with an appropriate cupriphore and presented in a medium with low water content.

A group of 29 re-anastomosed men were examined with respect to semen quality, anti-sperm antibody titres in serum and seminal plasma, presence of anti-sperm antibodies on sperm, and success rate in inducing pregnancy. Results indicated no association between pre-reversal serum anti-sperm antibody titres and post-reversal semen quality, but a pregnancy induction rate of zero was associated with serum anti-sperm antibody titres greater than 160. It is recommended that men considering reversal, with anti-sperm antibody titres of this level, should receive counselling about the possibility of post-reversal infertility.

The effects of cyclic adenosine monophosphate (cAMP), MnCl2, caffeine, and theophylline at concentrations of 10 mM on human sperm motility and forward migration in vitro were tested. cAMP was effective in activating human spermatozoal motility and forward migration after incubation with the spermatozoa for 3 hr or more and 5 hr, respectively, whereas caffeine and theophylline were effective in activating sperm motility after incubation with the spermatozoa for 1 hr or more. Caffeine and theophylline significantly activated sperm forward migration only after incubation with the sperm for 5 hr. MnCl2, a potent sperm adenylate cyclase activator, had no significant effect in improving human sperm motility and forward migration.

To eliminate the possibility of immunological infertility in spontaneously infertile and re-anastomosed men, a screening test that can be applied directly to semen is desirable. The SpermMar test is one such possibility. In this study, indirect tests for sperm antibodies using the commercial SpermMar test have been applied to a panel of sera whose reactions in the tube slide agglutination test (TSAT), gelatin agglutination test (GAT) and sperm immobilization test (SIT) for sperm antibodies are well characterized. The results from the SpermMar tests are compared directly with those obtained from Immunobead tests carried out at the same time. Results from screening tests performed on 30 sera confirmed complete correspondence between the GAT, SpermMar and Immunobead tests. When sera were titrated, the Immunobead test proved slightly more sensitive than the GAT and the SpermMar test was slightly more sensitive than the Immunobead test. The SpermMar test proved easier to use and to assess than the Immunobead test and it is recommended for consideration as a screening procedure for sperm antibodies despite the fact that at this stage only IgG antibodies can be detected.

Summary.The metabolism and motility of human ejaculated spermatozoa incubated in vitrowith steroids were studied. Progesterone and norethynodrel depressed the respiration, glycolytic metabolism and the motility of washed spermatozoa. Lynoestrenol did not affect the respiration or glycolysis of the spermatozoa, but did inhibit motility. Oestradiol did not cause any consistent alteration of the sperm metabolism, and did not affect the motility. Progesterone and norethynodrel appear to act on the plasma membrane of human spermatozoa to increase its permeability and hence to facilitate the loss of essential cofactors required for the glycolytic and oxidative processes.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.

Depletion of glutathione by inhibition of its synthesis by buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, leads to increased sensitivity to (i) irradiation and (ii) oxidative stress. In the present work, an intracellular cysteine delivery system was used to promote glutathione synthesis, and this was found to protect against toxicity. Thus, administration of L-2-oxothiazolidine-4-carboxylate protected against acetaminophen toxicity in mice; the thiazolidine, which is converted to L-cysteine by the enzyme 5-oxo-L-prolinase (present in many animal tissues and in plants) promotes the synthesis of glutathione, which is the actual protectant. The effect of this thiazolidine in increasing the level of glutathione is prevented by administration of buthionine sulfoximine. This thiazolidine may be useful in the treatment of other toxicities and in the treatment of certain diseases. It may also be valuable as a component of amino acid mixtures used in therapy and as a safener in agriculture.

Summary Saliva specimens have been examined electrophoretically, using polyacrylamide gel slabs. By slicing the stabs in halves, incubating one half-gel in starch followed by Staining with iodine and staining the other half-gel with amido black, amylase isozymes are visualized as bands of enzymic activity, up to 8 in number, 4 of which can usually be seen on amido black staining.Several different isozyme patterns have been observed. The results of pedigree studies suggest simple modes of inheritance of each of the variants. However, because different variants each involve only one or two of the isozymes, it appears that more than one structural gene locus is involved in the production of salivary amylase isozymes.Australian Journal of Experimental Biology and Medical Science (1969) 47, 97–103; doi:10.1038/icb.1969.9

While studying spermagglutinating sera, a number of sera which were positive in a microagglutination test (Franklin & Dukes, 1964), but which were negative in a macroscopic test in gelatin medium (Kibrick, Belding & Merrill, 1952) were found (Boettcher, Kay, Rümke & Wright, 1971). It appears that the agglutinins in these sera are not immunoglobulins (Boettcher & Kay, 1969; Boettcher, Hay, Kay, Baldo & Roberts, 1970). During procedures for fractionating these sera, much of the activity is often lost. One cause of the loss is dialysis. Sera, dialysed overnight against phosphate-buffered saline, have shown marked decreases in their spermagglutinating activities, though their haemagglutinating activities have been unaltered (Boettcher & Kay, 1969; Boettcher et al., 1970).Reported here are the results of experiments designed to determine whether the

Using a haemagglutination technique, heterologous antisera to both the seminal plasma and spermatozoa of man, possum, rabbit and ram and the whole homogenized epididymis and vas deferens of the rat, were found to show immunological cross-reaction with homologous erythrocytes. Cross-reaction between rabbit antiserum to rooster seminal plasma and chicken erythrocytes was observed, but cross-reaction was not seen with antiserum to rooster spermatozoa.Rabbit antiserum to bull seminal plasma haemolysed bovine erythrocytes, but haemolysis was not observed with antiserum to bull spermatozoa.The results obtained are discussed with particular reference to other papers reporting such cross-reacting antigens.

Human spermagglutinating sera showed three patterns in their reactions in a microagglutination (Franklin and Dukes, F and D) and a macroagglutination in gelatin (Kibrick) test. Some sera were active in the F and D test only; some were active in the Kibrick test only; and some were active in both. Eight sera of the first type, two of the second type, and three of the third type have been fractionated by zone electrophoresis, gel filtration in Sephadex G-200, and by ion exchange chromatography on DEAE-cellulose.Sera which were positive in only the F and D test were shown to have spermagglutinins which are nonimmunoglobulins. The agglutinins have a high mol wt and the electrophoretic mobility of β-globulins. Their activity appears to be directed towards seminal plasma, since they agglutinated fresh sheep red blood cells coated with human seminal plasma coupled by glutaraldehyde.Sera which acted in the Kibrick test were shown to possess spermagglutinins which were immunoglobulins. The sera which reacted by the Kibrick test only had IgG spermagglutinins. Other sera had IgG which reacted in both the Kibrick and F and D tests. One serum had only IgM agglutinins that agglutinated by both the Kibrick and F and D techniques. The reason for some sera agglutinating by only the Kibrick technique, whereas others agglutinated by both techniques, is uncertain, but possibly indicates different specificities of the sera.None of the human sera possessing spermagglutinating immunoglobulins agglutinated glutaraldehyde-treated fresh sheep red blood cells coated with seminal plasma. However, rabbit antisera directed against seminal plasma did. This suggests that the human antibodies are not directed at those components of seminal plasma which readily adhere to sheep red cells under the conditions used.

SummaryA-substance molecules were precipitated from salivas from A1and A2secretors with a Dolichos biflorus seed extract. The amounts of precipitate formed were shown to be proportional to the agglutination inhibition titres with the extract. It is concluded that both precipitating ability and inhibition titre can be taken as quantitative measures of antigen present in saliva.The decrease in Ulex europaeus (anti-H) and Dolichos biflorus (anti-A) inhibition titres of the salivas during the precipitation procedure was determined. The ratio of these values was calculated for the individual salivas, and was found to be higher for the A2than for the A1salivas. It is proposed that individual A-substance molecules from A1secretors have a higher ratio of A : H antigenic determinants than have those from A2secretors, and that one difference between the A antigens of A1and A2individuals, thus, has a quantitative basis. This conclusion forms part of the evidence leading to the proposal that the gene IA1is basically a duplication of the gene IA2.Australian Journal of Experimental Biology and Medical Science (1967) 45, 485–493; doi:10.1038/icb.1967.47

SummaryThe agglutination inhibition titres of salivas from secretors of blood groups A1, A2, B and O have been determined using human anti-A and anti-B sera, and Ulex europaeus and Dolichos biflors seed extracts. The mean inhibition titres of A1salivas with anli-A and Dolichos extract are significantly greater than those for A2salivas. The mean inhibition titres of salivas from individuals of different bloood group with Ulex extract decrease in the order O, A2, A1, B, which is noted as being different from the order for agglutination of blood cells of these groups by Ulex extract.Correlation coefficient for the titres of A1and A2salivas with anti-A and Ulex extract, and with Dolichos and Ulex extracts, are not significantly different from O, neither is the correlation coefficient for the titres of B salivas with anti-B and Ulex extract, whereas that for A1and A2salivas with anti-A and Dolichos extract is significantly O. It is concluded that the rates of production of antigenie factors controlled by the H and ABO loci are independent and that “aberrant secretors” are individuals in whom these rates differ greatly.Australian Journal of Experimental Biology and Medical Science (1967) 45, 495–506; doi:10.1038/icb.1967.48

SummaryThe distribution of the ability of saliva from 55 blood group A secretors to inhibit a human B antiserum was found to be bimodal. Subsequent determination of the subgroups of these people showed that the class with the smaller mode was associated with A substance in the salivas of A2secretors, the other with A substance in the salivas of A1secretors. In this study the two classes had non-overlapping distributions, though not in a larger subsequent one. Statistical evaluation of these results and of confirmatory tests on salivas from persons whose A subgroups had been previously determined have established that, on the average, salivas from group A1secretors have a greater capacity to inhibit a human anti-A serum than those from A2secretors.Australian Journal of Experimental Biology and Medical Science (1964) 42, 703–706; doi:10.1038/icb.1964.66

SCA has been identified as an iron-binding protein present in seminal plasma. It has an electrophoretic mobility similar to transferrin, and different from lactoferrin, whereas it shares immunological characteristics with lactoferrin not possessed by transferrin. The name scaferrin is proposed for this compound.

ABO blood group antigens on spermatozoa have been investigated using the agglutination inhibition technique. Antigens were not detected on spermatozoa from non-secretors, though they were detected on spermatozoa from secretors. Further, it has been demonstrated that spermatozoa will adsorb ABO antigens from aqueous solutions. The inhibition titres of seminal plasma and of spermatozoa from secretors were shown to be correlated.It is concluded that ABO antigens are found only on seminal spermatozoa from secretors and that these are adsorbed from the seminal plasma.

THERE is uncertainty about the chemical nature of spermagglutinins detected in the sera of some men and some women. The investigations described in this article gave results which appear to conflict with earlier suggestions that they are antibodies1or γ-globulins2and, in particular, IgG3. Preliminary studies in this laboratory, using ammonium sulphate fractionation of several spermagglutinating sera, had shown that the 60 per cent saturated fractions possessed the greatest spermagglutinating activity, whereas the greatest concentrations of γ-globulins were in the 40 per cent fractions4.

Washed spermatozoa from an O non-secretor have been incubated with seminal plasma from an A secretor and have been shown then to have developed the capacity of inhibiting anti-H and anti-A test solutions. This demonstration is in agreement with the findings of Edwards, Ferguson & Coombs (1964) who, in contrast to earlier workers, were unable to detect ABO blood group antigens on spermatozoa from non-secretors, though they were able to detect them on spermatozoa from secretor donors.

Some formerly unknown compounds with BNSi structure fragments such as (IV), (VI), (IX), (XI), (XIV), (XVI), (XVIII), and (XXI) are prepared according to the scheme.

Components from fasting human serum that could stimulate the forward migration of human spermatozoa were isolated by gel filtration on Sephadex G-25. The results suggest that these components may be used to enhance sperm forward migration and, hence, pregnancy rate in artificial insemination with husband's semen, (AIH) especially in cases where the sperm forward migration is not optimal.

Aging and clinical practice: depression and coexisting disease. R. Robinson, P. Rabins, eds. New York: IgakuShoin; Sydney: Butterworths 1989 (xii +242 pp., $55.00).