Expression and purification of the HIV type 1 Rev protein produced in Escherichia coli and its use in the generation of monoclonal antibodies.

We have developed a simple and rapid procedure for the purification of large amounts of Rev protein overexpressed in E. coli. The purification method, which does not require denaturation of the protein, takes advantage of the positively charged nature of Rev and the ability of Rev to interact with nucleic acids. The purified protein was used to develop three novel murine monoclonal antibodies against Rev. Using fusion proteins between glutathione S-transferase (GST) and various fragments of the Rev protein, we mapped the specificity of these antibodies to different regions of the Rev protein. One antibody, 3H6, is directed against the nucleolar localization/RRE-binding domain of Rev between amino acids 38 and 44. Another antibody, 3G4, recognizes an epitope between amino acids 90 and 116 of Rev. A third antibody, 2G2, does not recognize any of the fusion proteins, and may be directed against a conformational epitope. All three antibodies are able to detect Rev on Western blots and to immunoprecipitate Rev under native conditions. However, only 3H6 and 3G4 immunoprecipitate Rev under denaturing conditions and are able to detect Rev expressed in transfected cells by indirect immunofluorescence. These antibodies should prove useful in further studies of Rev function.

Signatures of the martian atmosphere in glass of the Zagami meteorite.

Isotopic signatures of nitrogen, argon, and xenon have been determined in separated millimeter-sized pockets of shock-melted glass in a recently identified lithology of the meteorite Zagami, a shergottite. The ratio of nitrogen-15 to nitrogen-14, which is at least 282 per mil larger than the terrestrial value, the ratio of xenon-129 to xenon-132 = 2.40, and the argon isotopic abundances match the signatures previously observed in the glassy lithology of the Antarctic shergottite EETA 79001. These results show that the signatures in EETA 79001 are not unique but characterize the trapped gas component in shock-melted glass of shergottites. The isotopic and elemental ratios of nitrogen, argon, and xenon closely resemble the Viking spacecraft data for the martian atmosphere and provide compelling evidence for a martian origin of the two shergottites and, by extension, of the meteorites in the shergottites-nakhlites-chassignites (SNC) group.

Mapping of an assembled epitope of human follicle-stimulating hormone-beta utilizing monoclonal antibodies, synthetic peptides, and hormone-receptor inhibition.

Monoclonal antibodies (mabs) to human (h) FSH were utilized to probe epitopes of the beta-subunit of hFSH (hFSH beta). These mabs had an average approximate affinity constant (Ka) of 10(8) M-1 for hFSH beta and 10(7) M-1 for heterodimeric hFSH. Hormone specificity of mabs for hFSH beta was demonstrated by a lack of cross-reactivity with hCG alpha, FSH alpha, or LH alpha. Epitope specificity of each mab was initially assessed by determining whether solid phase mab could bind to [125I]hFSH already bound to mabs in liquid phase. In addition, it was determined whether [125I]mab could bind to hFSH already bound to solid-phase mabs. Both epitope cross-matching protocols indicated that all mabs bound to the same epitopes on hFSH beta. Next, synthetic peptides corresponding to the sequence of hFSH beta were used in an enzyme-linked immunosorbent assay to map this epitope. All mabs bound to peptides 7-19, 1-20, 33-53 and 66-85 but did not bind or bound weakly to peptides 81-100, 95-103, and 103-110. Titration experiments were performed using different concentrations of peptide (0.3-41 nmol) and one mab 3G3 (500 ng-25 ng) in the enzyme-linked immunosorbent assay. The product of the lowest mass of both peptide and antibody which gave a positive result was used to rank the peptides for their binding with mab 3G3. Peptides were ranked in the following descending order of potency: 33-53, 49-67, 66-85 much greater than 16-36, 1-20, 95-103, 52-65, 81-100, and 103-110. Ability of the mabs to inhibit binding of [125I]hFSH to bovine testis membrane receptor (Rec) was also studied. When [125I]hFSH was preincubated with increments of each mab for 2 h at 25 C before adding Rec with further incubation for 16 h, all mabs inhibited [125I]hFSH binding to Rec. The data suggest that most of the hFSH beta molecule has a conformation enabling all antibody recognizable regions to be in close proximity to each other. The present study provides evidence for an assembled epitope comprising in part, amino acids 33-53, which has been previously shown to be involved in receptor binding. Peptide sequences 49-67 and 66-85 are neighboring sequences in this assembled epitope which contains the determinants for receptor binding.

A rat granulosa cell plasminogen activator bioassay for FSH in human serum.

A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3.5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0.3-15 IU/l, with an assay sensitivity of 0.3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 degrees C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 microliters) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0.745, n = 15 and P less than 0.05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples.

Negative feedback of secretion of follicle-stimulating hormone by the pituitary gland of developing male rats.

Rabbit antiserum to human seminal plasma inhibin (hSPI) was administered subcutaneously to developing male rats of 5, 10, 14, 17 and 24 days of age and the size of the endogenous FSH rise in serum was measured. The FSH levels were threefold higher on day 9 and 1.5-fold higher on days 14 and 18 when compared with levels in control rats treated with normal rabbit serum. Furthermore, the in-vitro binding capacity of pituitary plasma membrane to 125I-labelled hSPI declined with increase in age of the rats. Thus, the results of the present study suggest that the sensitivity of the testicular inhibin-FSH feedback relationship is related to age-dependent changes in pituitary binding of inhibin.

Levels of inhibin in human semen and accessory reproductive organs.

Levels of inhibin in the seminal plasma of oligozoospermic and normozoospermic semen and normal human accessory reproductive organs were estimated using specific and sensitive radioimmunoassay (RIA). Seminal inhibin content showed a positive correlation (r = 0.827) with the sperm concentration. Further, in the human accessory reproductive organs, prostate showed maximum content of inhibin as compared to the seminal vesicle and the epididymis.

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.

Alteration in testicular cyclic AMP system in the rat following vasectomy.

Endogenous cyclic AMP levels and the activities of adenylate cyclase, cyclic AMP-dependent and independent protein kinases were examined in testes of mature rats bilaterally vasectomized for one, three and seven months. Although no significant alteration in testicular cyclic AMP was detected one month following vasectomy, marked decreases (by 55% and 32%, respectively) were seen three and seven months postvasectomy. Likewise, vasectomy also resulted in a significant decrease (by 25%) in the activity of testicular adenylate cyclase three and seven months after vasectomy. Although soluble cyclic AMP-dependent protein kinase activity remained unaffected three months postvasectomy, the activity of the cyclic nucleotide-dependent enzyme was significantly increased (by 21%) when compared to the sham-operated controls. Furthermore, while the protein kinase ratio (--cyclic AMP/+cyclic AMP) was decreased in animals vasectomized for three months, the ability of the enzyme to bind (3H) cyclic AMP in vitro was significantly enhanced (18%). Rats vasectomized for seven months showed similar biochemical alterations but the effects of this procedure were more pronounced. Moreover, while short-term vasectomy increased the responsiveness of seminiferous tubular adenylate cyclase to in vitro stimulation by follicle stimulating hormone, the activity of the enzyme was also increased (by 100%) in the presence of luteinizing hormone in vasectomized rats. These data raise the possibility that changes in testicular function seen following vasectomy may be related to the alterations in cyclic AMP metabolism as well as in the sensitivity of testicular adenylate cyclase to regulation by gonadotropins.

Sequential changes in hepatic polyamine, deoxyribonucleic acid, and cyclic adenosine 3',5'-monophosphate metabolism after subacute exposure to cadmium in rats.

Heavy metal treatment (2 X 1 mg/kg per day) for 3, 5, and 7 days resulted in progressive augmentation in the incorporation of [14C]thymidine into hepatic DNA. In contrast with the observed enhancement in DNA synthesis, cadmium exposure tended to produce a decrease in the activity of hepatic ornithine decarboxylase (EC 4.1.1.17) at 1, 3, or 5 days with the lowest (34% of control values) enzymic activity seen after 7 days. A similar reduction in the activity of S-adenosylmethionine decarboxylase (EC 4.1.1.50) was observed in livers of rats treated with cadmium for 1-7 days. Subacute exposure to cadmium significantly lowered the hepatic levels of spermidine and spermine whereas the endogenous concentrated of putrescine remained unaltered. In addition to the observed effects on the biosynthesis of polyamines and DNA, heavy metal treatment produced stimulation of the hepatic adenylate cyclase (EC 4.6.1.1)--cyclic AMP system. Significant increases in the activity of hepatic adenylate cyclase and endogenous cyclic AMP levels were detected as early as 1 day and the observed alterations persisted during the entire 1-week period of cadmium exposure. The depression in polyamine formation was accompanied by enhanced DNA biosynthesis as well as stimulation in the adenylate cyclase-cyclic AMP system of rat liver.

Effect of administration of a single dose of testosterone oenanthate on constituents of human seminal plasma and serum gonadotropins.

Testosterone oenanthate was administered intramuscularly in six infertile men with oligozoospermia and its effects on serum gonadotropins and some constituents in the seminal plasma were studied. One week after injection the mean serum FSH level was decreased to about 50%. Serum LH levels did not change. The mean ornithine decarboxylase activity in human semen was increased by 100% after the testosterone administration. The androgen dependent nature of ODC, fructose and sialic acid have been demonstrated.

PIP: The effect of a single im injection of testosterone enanthate (TE) on serum gonadotropins and constituents of seminal plasma was studied in 6 oligospermic men. Within 1 week of injection, mean serum follicle stimulating hormone (FSH) levels were reduced by 50%, while luteinizing hormone (LH) levels were unaltered. Treatment increased the mean ornithine decarboxylase activity in seminal plasma by 100%. The citric acid content and maltase activity of seminal plasma were not markedly altered. Fructose concentrations were significantly (p less than .05) higher during the 3rd and 4th weeks after injection, while sialic acid concentrations were significantly (p less than .05) elevated during the 2nd week. The results indicate that ornithine decarboxylase activity in seminal plasma may be a useful indicator for evaluating the secretory function of the accessory sex glands.

Antisera to gonadotrophins and placental function in mice. I. Effect on polyamine content.

The effect of antisera to ovine LH and ovine prolactin was studied on polyamine levels in the mouse placenta. The antisera were administered on Day 11, 12 or 13 of pregnancy and the mice were killed 24 hr later. The polyamines (spermine, spermidine and putrescine) in the placentae were estimated. Polyamine levels were reduced after treatment with anti-LH on any of the 3 days and after treatment with anti-prolactin serum on Days 11 or 12. Only the spermidine content was reduced when anti-prolactin serum was injected on Day 13 of pregnancy. Placental DNA and RNA levels paralleled those observed for polyamine content. The changes in polyamine content and nucleic acid levels indicate that these antisera to LH and prolactin interfere with placental function.