Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.     

The treatment of the hepatorenal syndrome with intra-renal administration of prostaglandin E1

Three patients with the hepatorenal syndrome were treated with prostaglandin E₁ administered through a selective renal arterial catheter. Prostaglandin E₁ was given in progressively increasing doses (2 to 100 ng/kg/min) over a 60-minute period. Control plasma prostaglandin E levels were elevated in all three patients, 0.98, 0.91, and 0.83 ng/ml, respectively. At the end of the infusion, plasma prostaglandin E levels had risen to 10.4, 2.63, and 10.3 ng/ml in the three patients respectively. Plasma renin activity increased during the course of the infusion in two of the patients. The plasma aldosterone concentration did not change during the prostaglandin E₁ infusion. Intrarenal prostaglandin E₁ failed to increase urine volume or urinary sodium concentration in three patients with the hepatorenal syndrome.     

The protective effect of dietary fish oil on murine lupus

Dietary marine lipids markedly reduce the severity of glomerulonephritis and its associated mortality in inbred strains of mice developing autoimmune disease, a model for human systemic lupus erythematosus. We report here the influence of varying the dose of menhaden oil and the timing of its administration on the mortality of female (NZB x NZW) F₁ mice. After ingesting 25 wt% menhaden oil (MO) for periods of 1.5 weeks to 12 months, there was a stable content of tissue n-3 fatty acids, with total n-3 fatty acids of 28% and 35% in spleen and liver, respectively. The extent of protection from mortality was dependent on the dose of MO with marked protection at doses of 11 to 25%, marginal protection at 5.5% and no protection at 2.5% MO. Delay in the institution of MO until ages 5 or 7 months still resulted in large reductions of mortality. Conversely, institution of a MO diet from 6 weeks until ages 5 to 7 months followed by a change to beef tallow resulted in little protection. Serum levels of 4 cyclooxygenase products were reduced ranging from 26 to 76% in mice fed MO diets, compared to mice fed beef tallow, based on radioimmunoassay. The degree of reduction of mortality on different doses of MO was correlated best with tissue levels of C22:5, and levels of C20:5 and C22:6 were similar at high and low doses of MO, suggesting that levels of 22:5 may be related to the protective effects of marine lipids on autoimmune disease.     

Zymosan activated plasma infusion in sheep: Effects of ethanol administration

Zymosan activated plasma infusion induces pulmonary sequestration of neutrophils and the release of TXA₂ into the pulmonary vascular bed causing profound and transient pulmonary hypertension.Since ethanol (ETOH) inhibits several inflammatory functions of neutrophils, including adherence and aggregation, we examined the ability of anesthetic doses of ETOH to alter the hemodynamic and cellular response to the infusion of zymosan activated plasma (ZAP) in vivo. Twenty five ml of autologous ZAP was intravenously infused into five control and seven (ETOH-treated sheep during mechanical ventillation. In control sheep the mean pulmonary artery pressure (PAP) transiently increased from 14.7±1.4 mm Hg (mean±SEM) to a pead of 38+8 mm Hg by three minutes after beginning the infusion of ZAP. Blood leukocyte concentration transiently decreased 19% below the baseline value due to pulmonary sequestration of polymorphonuclear leukocytes (PMN). Plasma TXB₂ levels measured by radioimmunoassay (RIA) increased from 0.2 to 5.4 ng/ml six minutes after the initiation of ZAP infusion.In five sheep, intravenous infusion of 200 ml of 96% ETOH yielded very high plasma concentrations (882±101 mg%) and completely inhibited both the rise of PAP and the increase of plasma TXB₂ levels after ZAP infusion. However, blood leukocytes transiently decreased 58% below the baseline value. Lower plasma levels of ETOH (200 and 400 mg%) did not prevent either the increase of PAP or the elevation of plasma TXB₂ after ZAP infusion.     

Thromboxane and pulmonary hypertension following E. coli endotoxin infusion in sheep: Effect of an imidazole derivative

We assessed the effect of a specific thromboxane synthetase inhibitor (an imidazole derivative) on pulmonary hemodynamics and the concentrations of TxB₂ (TxA₂), 6-keto-PGF_1α (PGI₂), and PGF_2α in pulmonary lymph and transpulmonary blood samples following intravenous administration of E. coli endotoxin (1 μg/kg) in sheep. In control animals the rise in pulmonary artery pressure correlated with increases in plasma and lymph TxB₂ concentrations and large transpulmonary concentration gradients of this metabolite were measured. In imidazle treated animals both pulmonary hypertension as well as increases in plasma and lymph TxB₂ concentrations were substantially reduced. In contrast, peak concentrations of 6-keto-PGF_1α (PGI₂) and PGF_2α were severalfold higher than those measured in control animals. This suggests a shunting of endoperoxide metabolism towards prostacyclin and primary prostaglandins and documents the specificity of the thromboxane synthetase inhibitor. Out study provides evidence that endotoxin-induced pulmonary hypertension is mediated by pulmonary synthesis of TxA₂.     

Group mating among Norway rats II. The social dynamics of copulation: Competition,cooperation, and mate choice

The social interactions within groups of Norway rats (Rattus norvegicus) had a strong impact on the individual pattern of copulation which, in turn, affects sperm precedence and the probability of implantation in this species. Males alternated uninterrupted ejaculatory series, augmenting each others' copulatory investment. Females took turns mating after receiving an intromission, collectively potentiating the males' copulatory behaviour; increasing the number of oestrous females increased the number of intromissions and ejaculations achieved by each male but did not affect the amount of copulation experienced by each female. These turn-taking patterns within each sex provided the opportunity to change partners and permitted the emergence of different sex-typical patterns of copulation. Furthermore, the dominant male contributed more intromissions and tended to give each female more ejaculations than the subordinates did. Dominant males were also more likely to inhibit the subordinates' sperm transport. Females competed among themselves for the opportunity to mate with a male as he approached ejaculation and were likely to protect more of the dominant male's sperm transport than the subordinate male's.     

Cell polarity in precartilage mouse limb mesenchyme cells

The patterns of orientation of individual mesenchyme cells have been evaluated in the hindlimb of the mouse embryo during the period of transition from early aggregation (Day 12) to cartilage formation (Day 13). Orientation was measured by determining the angular relationship between the Golgi-nucleus axis of each cell relative to either the longitudinal limb axis or the center of the cartilaginous aggregate. Patterns were assessed qualitatively and quantitatively in horizontal, vertical, and transverse sections of the proximal, middle, and distal precartilage mesenchyme. These analyses showed that the mesenchyme cells are oriented predominantly toward the longitudinal axes of both the early (Day 12) and late (Day 13) aggregates.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Conversion of methionine to homocysteine thiolactone in liver

Since hemocysteinemia is associated with arteriosclerosis, the conversion of methione to homocysteine thiolactone was studied in guinea pig liver in vivo. 60 min after intraperitoneal injection of [¹⁴C]methione, [¹⁴C]homocystein thiolactone was found to constitute 9.1% ± 0.2 of the lipid bound ¹⁴C and 20% ± 1.0 of the acid soluble ¹⁴C. This conversion is the first step of a new pathway by which the sulfur of methionine is transferred to phosphoadenosine phosphosulfate.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Group mating among Norway rats I. Sex differences in the pattern and neuroendocrine consequences of copulation

The copulatory pattern of groups of rats (Rattus norvegicus) was studied in the laboratory in a seminatural environment. In a given mating session, every oestrous female copulated with each male; likewise, every male copulated with each oestrous female. While individual males and females experienced similar amounts of copulation, there were dramatic sex differences in sequence and temporal pattern. Males mated in a multiple intromission pattern and had more ejaculatory series when several females were in oestrus. In contrast, females received intromissions and ejaculations in a random order, not in the sequence of a male ejaculatory series. Males copulated at shorter intervals than females did, a temporal sex difference that was determined by the pattern of female solicitations and male approaches. These sex differences are used to discuss the different units of analysis that are appropriate for male and female sexual behaviour in this species. Furthermore, the sex differences in the temporal pattern of copulation which emerged during group mating parallel the known sex differences in the temporal parameters of the neuroendocrine reflexes which mediate successful reproduction in the domestic strain.     

On the biochemical similarities of ascorbic acid and interferon

The anti-viral effects of ascorbic acid and interferon are proposed to result from the peroxy radical mediated biosynthesis of prostaglandins and from the direct attack of free radical species on unsaturated lipids in the cell membrane. These processes are proposed to lead to changes in fluidity of the host cell membrane which inhibit the fusogenicity of the attacking virus.     

Patterns of cell polarity in the developing mouse limb

Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are exreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

The Schistosoma haematobium egg granuloma

The etiology of the granulomatous response around Schistosoma haematobium eggs in mice was investigated. Eggs injected into the microvasculature of the lungs of mice evoked a granulomatous reaction which was demonstrated at 8 and 16 days. Prior exposure of mice to the eggs or to crude soluble egg antigens (SEA) resulted in significantly larger granulomas than in the unsensitized controls. The degree of sensitization was dependent both on the route of administration and the quantity of eggs. Furthermore, this phenomenon could be adoptively transferred with spleen cells from previously sensitized mice but not with serum. Mice sensitized with S. haematobium eggs and challenged by injection of SEA into their footpads developed both immediate and delayed swelling. Cross-sensitization between S. haematobium, S. mansoni and S. japonicum eggs was also studied in both the lung and footpad systems.     

Helper factor(s) augment in vitro induction of tumor-specific immunity

Helper factor supernatants derived from alloantigen-activated murine lymphocytes augment the generation of cytolytic effector cells to syngeneic tumor cells. The effects are dose dependent and vary with the syngeneic tumor cell system studied. The effector cells are specific for the tumor-associated antigen(s) utilized for their induction, and are sensitive to lysis with anti-T-cell serum (Thy 1.2), but are insensitive to lysis with an allogeneic anti-NK-cell serum. The helper factor supernatants also augment the production of a “tissue-culture-induced” cytolytic cell (cultured NK cell), which is resistant to treatment with both anti-Thy 1.2 and anti-NK serum.     

Long-term skin preservation by combined use of tissue culture and freezing techniques.

Simple but effective methods for shipping newly excised rabbit skin to a distant central laboratory for in vitro culture on a pigskin base, followed by freezing in a cryoprotective agent (DMSO) for frozen storage and subsequent reshipment to the originating laboratory while still frozen are described. At a suitable time the frozen tissue was rapidly thawed and transplanted to the autologous recipient rabbit. Of 12 cultures, seven indicated good to excellent cell growth and maturation on the host. The successful method combined the use of in vitro tissue culture and freezing to permit a central laboratory to grow the skin and ship it to the originating center for autografting at a convenient time.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.