Splitting of wheelrunning rhythms by castrated or steroid treated male and female hamsters

Male and female hamsters, with or without gonadal hormones, were housed in constant light (LL) while wheel running rhythms were recorded. Estradiol benzoate (EB) in Silastic capsules reduced rhythm desynchronies, such as splitting, in ovariectomized animals compared to blank implanted controls. In males, there were no significant effects of testosterone or EB in Silastic implants, castration or sham operation on incidence of rhythm desynchronies. Males generated split rhythms which differed from females in clarity and the angle at which the limbs of the splitting rhythms diverged. Other differences were (a) greater activity onset variability for castrated females with relatively little onset variability for other groups and (b) more running time by EB treated males than any other group. Splitting for all animals occurred with an average latency in LL of 55±3 days; the period stabilized in 12±1 days and was 0.2 hr shorter in length. The two limbs of the split rhythm were a mean 181±5° apart. Induction of splitting by LL is critically discussed with special reference to the two oscillator model of hamster activity and existing evidence for more than two oscillations in wheelrunning activity.     

Effect of cycloheximide on the distribution of alpha-naphthylisothiocyanate in rats

Others have shown that in rats the administration of cycloheximide (2 mg/kg) within 2 hr before or after 300 mg/kg of α-naphthylisothiocyanate (ANIT) blocks the development of hyperbilirubinemia and cholestasis. The effects of cycloheximide on the tissue distribution of ANIT labeled with ¹⁴C or ³H were examined. In cycloheximide-treated animals, less ANIT-derived radioactivity was found in blood, liver, fat and several other tissues at various times after ANIT than in controls. Rats given a mixture of (naphthyl-4-³H) ANIT and (isothiocyanate-¹⁴C) ANIT with a ratio of ${}^6\text{H}{}^{14}\text{C}\text{:6.3}$, excreted in their bile during the first 8 hr after ANIT, more ³H compared to ¹⁴C. In rats treated with cycloheximide, the ${}^3\text{H}{}^{14}\text{C}$ ratio did not change. The data suggest that normal animals excrete an ANIT metabolite in their bile from which the ¹⁴C label in the isothiocyanate moiety has been lost, whereas this does not happen in cycloheximide-treated animals. This provides further evidence that biotransformation of ANIT is necessary for the development of hyperbilirubinemia and cholestasis. Bile might be a convenient starting material for the isolation and identification of ANIT metabolites.     

Formation of the guanylylated and methylated 5′-terminus of vaccinia virus mRNA

mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-³H]methionine and α- or β,γ-³²P-labeled ATP or GTP. The two 5′-terminal structures m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m were isolated after P₁ nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the ³²P-labeling data as well as by the formation of m⁷G(5′)ppp(5′)A^m- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m⁷G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered:

${}^{\mathrm{\gamma \beta \alpha }}\text{ppG}\text{+}{}^{\mathrm{\beta \prime \alpha \prime }}\text{ppN}\text{?→}\text{G}\text{(5′)}\text{N}\text{?+}{}^{\mathrm{\gamma \beta }}\text{PPi}$

$\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoHcy}$

$\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}{}^{\mathrm{m}}\text{+}\text{AdoHcy}$

     

Influence of exercise modality on agreement between gas exchange and heart rate variability thresholds

The main purpose of this study was to investigate the level of agreement between the gas exchange threshold (GET) and heart rate variability threshold (HRVT) during maximal cardiopulmonary exercise testing (CPET) using three different exercise modalities. A further aim was to establish whether there was a 1:1 relationship between the percentage heart rate reserve (%HRR) and percentage oxygen uptake reserve (%V˙O2 R) at intensities corresponding to GET and HRVT. Sixteen apparently healthy men 17 to 28 years of age performed three maximal CPETs (cycling, walking, and running). Mean heart rate and V˙O2 at GET and HRVT were 16 bpm (P<0.001) and 5.2 mL·kg^-1·min^-1 (P=0.001) higher in running than cycling, but no significant differences were observed between running and walking, or cycling and walking (P>0.05). There was a strong relationship between GET and HRVT, with R² ranging from 0.69 to 0.90. A 1:1 relationship between %HRR and %V˙O2 R was not observed at GET and HRVT. The %HRR was higher during cycling (GET mean difference=7%; HRVT mean difference=11%; both P<0.001), walking (GET mean difference=13%; HRVT mean difference=13%; both P<0.001), or running (GET mean difference=11%; HRVT mean difference=10%; both P<0.001). Therefore, using HRVT to prescribe aerobic exercise intensity appears to be valid. However, to assume a 1:1 relationship between %HRR and %V˙O2 R at HRVT would probably result in overestimation of the energy expenditure during the bout of exercise.     

Effects of two-hour light-dark cycles on feeding, drinking and motor activity of the rat

The effect of two-hour light-dark cycles on feeding, drinking and motor activity in the rat was compared with behavior under the usual $\text{12}\text{12}$ hour cycle. The two-hour cycles consisted of $\text{60}\text{60}$ min, $\text{80}\text{40}$ min and $\text{40}\text{80}$ min light-dark schedules which were maintained each for 7 days. Water intake, frequency of feeding, and motor activity were still significantly higher during dark than during light, although their occurrence during dark was reduced as compared to the $\text{12}\text{12}$ hour control schedule. A free-running circadian rhythm of consummatory behavior with a period length exceeding 24 hours was present throughout the experimental period. The amplitude of the circadian feeding rhythm gradually decreased over time, whereas the percentage of feeding during dark increased. During the circadian phase of minimal food intake, illumination changes affected feeding behavior more strongly than during the phase of maximal food intake. After restoration of the orginal $\text{12}\text{12}$ hour cycle, the amplitude of the nocturnal feeding rhythm increased gradually over several days, whereas the amplitude of the drinking rhythm showed a more rapid recovery. The experiments show that even short cycles of illumination may exert control over the rat's consummatory and motor activity. Short light-dark schedules provide a way for studying separately effects of illumination and of circadian rhythms.     

Effects of steroids and gonadotropins on follicular development in the hypophysectomized hamster

To determine the rate of follicular development in long-term hypophysectomized (H ) hamsters, single IP injections of ³H-thymidine were given six days after H and autoradiographs were prepared from animals killed on days 6–15. Only follicles in stages 1 (2–3 layers of granulosa cells), 2 (4–5 layers) and 3 (6–7 layers) were present and there were fewer follicles than in intact, cyclic hamsters. The entire population of follicles in stages 1 and 2 was replaced every five to six days in hypophysectomized hamsters, whereas in cyclic hamsters, complete turnover takes eight days (Chiras and Greenwald, '77). Other groups of H hamsters were treated on days 4–6 with either estradiol benzoate (EB — 10 μg), progesterone (P — 1 mg) or EB and P and then given ³H-thymidine at 0900 hours of day 6 and killed one hour later. Steroid treatment affected early stages of follicular development: EB stimulated growth of these follicles; P alone had little effect; given with EB, P acted as an antagonist. Other H hamsters were treated with FSH (100 μg), LH (10 μg), FSH + LH, PMS (30 IU) or saline vehicle at 0900 hours six days post-hypophysectomy. They were injected with ³H-thymidine at 2100 hours on day 6 and killed one hour later. LH- and saline-treated animals had approximately the same number of follicles; however, there were no stage-3 follicles in the LH-treated group and the percentage of labelled follicles in stages 1 and 2 was significantly lower after LH treatment. FSH enhanced follicular development and thymidine uptake. Twice as many stage-3 follicles were seen in FSH-treated animals as controls; stage-4 follicles (>8 layers of granulosa cells) were also encountered after FSH treatment. Labelling Index (LI) and Intensity (L. Int.) for the FSH-treated group were the highest of all treatments. LH antagonized some of the effects of FSH. PMS resulted in follicular growth similar to that achieved by FSH; however, PMS markedly depressed LI and L. Int., which was probably due to the LH-like component of PMS. These results demonstrate that the gonadotropins affect the development of small follicles. FSH stimulates growth; LH depresses follicular growth when administered alone and antagonizes some of the effects of FSH. Hamsters pretreated with EB or P on days 4–6 and then given a single, subcutaneous (SC) injection of FSH at 0900 hours on day 6, were treated with ³H-thymidine at 2100 hours on day 6 to assess the interaction of steroids and FSH. EB pretreatment caused the development of stage-5 follicles (early antral follicles) but did not increase thymidine uptake (measured by LI and L. Int.) in the small follicles above that attained by FSH alone. P pretreatment diminished thymidine uptake in small follicles, but did not reduce their number. These results demonstrate that in the hamster the population of small follicles, i.e., the often misnamed “pituitary-independent” follicles, can be influenced quantitatively and qualitatively by steroids and gonadotropins.     

Daily and hourly estrous running in intact, spayed and estrone implanted rats

Daily and hourly running rates of spayed female rats implanted with estrone or injected with estradiol benzoate (EB) were examined to determine whether estrous periodicity in activity occurs in the absence of hormone fluctuations. No evidence of a 4–5 day estrous cycle was found in the estrone implanted or in the EB injected gonadectomized females. Periodicity did not appear when amplitude of running was reduced by injections of the antiestrogen, MER-25. The major difference in hourly rates of running found on the 4 days was that intact animals took fewer rests during the latter part of the night when in estrus. Estrone implanted animals' hourly running pattern did not vary over days. It was concluded that hormonal fluctuation rather than intrinsic neural rhythmicity was the major determinant of estrous running periodicity in the rat.     

Immunological recognition of cuticular transplants in insects

Transplantation of allogeneic and xenogeneic cuticle onto the American cockroach, $\text{Periplaneta americana}$, was carried out in order to compare the specificity of immune recognition of these ‘skin grafts’ with that of implanted tissues. In order to facilitate interpretation of results, the technique of transplanting cuticle from nymphal donors onto nymphal recipients was adopted - if donor subcuticular epidermis is not recognised as ‘foreign’, it will grow, fuse with the recipient's epidermal sheet and will be stimulated by the recipient's hormonal signals to produce new cuticle of donor type at the next moult. Neither allogeneic cuticle nor xenogeneic cuticle from $\text{Blatta orientalis}$ were recognised as foreign by the immune system of $\text{P. americana}$ - dark patches of $\text{Blatta}$-type cuticle were produced at the graft site post-moult. Conversely, xenogeneic cuticle of $\text{Blaberus craniifer}$ was not visible post-moult. These results corroborate those from implantation studies, that allogeneic tissues from $\text{P. americana}$ and xenogeneic tissues from $\text{B. orientalis}$ are immunologically compatible with $\text{P. americana}$, whereas xenogeneic tissue from $\text{Blaberus craniifer}$ is incompatible. Whether this incompatibility is immunological or ‘positional’ has not yet been determined; the observation that xenografts from $\text{Nauphoeta}$$\text{cinerea}$ do not reappear on $\text{P. americana}$ post-moult, whereas 50% of $\text{N. cinerea}$ implants are not recognised as ‘foreign’, suggests that ‘positional incompatibility’ (i.e. the signals responsible for formation of cuticular pattern are incorrect for the donor epidermis) may also play an important part in the rejection of $\text{N. cinerea}$ cuticular grafts.     

Mitogenic responses of frog lymphocytes to crude and purified preparations of bacterial lipopolysaccharide (LPS)

We have compared $\text{in}$$\text{vitro}$ mitogenic responses of frog ($\text{Xenopus}$$\text{laevis}$ and $\text{Rana}$$\text{pipiens}$) lymphocytes to various preparations of lipopolysaccharide (LPS). Commercial LPS prepared from $\text{E}$. $\text{coli}$ (phenol extraction) and from $\text{S}$. $\text{abortus-equi}$ (phenol and TCA extraction procedures) was mitogenic for frog lymphocytes. After reextraction of these LPS preparations with phenol-water, the remaining LPS was either considerably less mitogenic or not mitogenic. Purified $\text{E}$. $\text{coli}$ 055:B5 LPS, prepared by phenol water extraction, enzyme treatment and column chromatography, was not mitogenic. Frog cells proliferated poorly or not at all with all concentrations of reextracted or purified LPS tested (0.5–400 ug/ml) and at all culture periods examined (days 1–7). All LPS preparations used were mitogenic for CAF₁ mouse lymphocytes, whereas reextracted and purified LPS preparations were not mitogenic for lymphocytes from C3H/HeJ cells. $\text{Xenopus}$ were also not susceptible to toxicity induced by parenterally administered LPS in concentrations which killed CAF₁ mice.     

Response to NaCl taste in mixture with sucrose by sodium deficient rats

Sodium deficient, adrenalectomized rats and nondeficient control rats were offered, for 20 min, either a mixture of 0.15 M sodium chloride and 0.3 M sucrose, or a 0.3 M sucrose solution. The sodium deficient rats drank about 3 times more of the mixture than of the sucrose alone. Nondeficient control animals showed no differential preference for the mixture over the sucrose solution. Subsequent tests indicated that the amount of mixture ingested by the sodium deficient group was comparable to the intake of a much weaker ($\text{1}\text{5}$ as strong) sodium chloride concentration given alone. These results are discussed in the context of taste component analysis of mixtures and suggest that the rodent taste system can specificially respond to sodium chloride in a sodium chloride-sucrose mixture     

Inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid

Eighteen derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid were assayed for inhibitory activity against neuraminidases from viral and bacterial sources. Twelve of these compounds were active against neuraminidases of Vibrio cholerae, influenza $\text{A}\text{Mel}\text{,}\text{B}\text{Lee}$, and Newcastle disease virus, causing 50% enzyme inhibition in concentrations ranging from 10^?3M to 10^?6M. The most active of them and the most potent neuraminidase inhibitor described so far is 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This compound has an inhibitor constant (K_i) of 7,9 × 10^?7, M for influenza $\text{A}\text{Mel}$ virus neuraminidase whereas the K_m of the virus enzyme for the substrate is 1000 times weaker (K_m = 6, 9 × 10^?4M). The mechanism of inhibition is competitive, and enzyme inhibition is independent of enzyme concentration. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid inhibits hemagglutination by NDV and SV5 but does not inhibit agglutination of red cells by Sendai virus or influenza A and B viruses.     

Endocytosis by echinoid phagocytosis in vitro. I. Recognition of foreign matter

Endocytosis, with emphasis on phagocytic recognition was examined in the sea urchin $\text{Strongylocentrotus droebachiensis}$. Most cells (about 67%) in the coelomic fluid were phagocytes. There was also a linear uptake of colloidal gold with time in the cultured cells, presumably caused by pinocytosis. No opsonizing activity was found in the cell-free coelomic fluid, and the parallel phagocytic avidity $\text{in vivo}$ and $\text{in vitro}$ of different particles also indicates that humoral factors were absent or not essential for recognition of foreign surfaces by phagocytes. Untreated erythrocytes (RBC) were taken up at a low rate, while RBC treated with aldehyde, Fe²⁺, tannin, Con A, or IgM + mouse C5-deficient complement were phagocytosed efficiently. Phagocytosis was reduced when RBC were covered with specific antibodies. Thus, the echinoid phagocyte lacks an Fc receptor, but may have a C3b receptor on its membrane. Other particles with high phagocytic avidities were carbon, Sephadex, latex and $\text{Escherichia coli}$. Thus the cell membranes of phylogenetically distant phagocytes may exhibit common structural features responsible for foreign surface recognition.     

Tail pinch-induced eating does generalize to a nonpreferred but familiar food.

Mild tail pinch has been reported to induce many of the same behaviors as electrical stimulation of the brain. In this study a 4 min tail pinch induced significant eating of either canned dog food or rat chow. In contrast to eating produced by brain stimulation, which does not generalize along the appropriate stimulus dimension of food objects, removal of the tail pinch-induced eaters' preferred food resulted in their eating another nonpreferred, but familiar food. With only the nonpreferred food available, intake of animals who preferred rat chow increased and intake of animals who preferred dog food decreased compared to their respective controls, although no initial differences existed. When both foods were again available, $\text{13}\text{18}$ animals retained their initial preference while $\text{5}\text{18}$ showed no preference. No animals reversed their preference. Tail pinch-induced eating may be more similar to normal eating than that produced by electrical brain stimulation.     

Cytotoxic testing for food allergy: Evaluation of reproducibility and correlation

Cytotoxic food tests still present conflicting opinions concerning their validity. Nine atopic patients with or without a history of food allergy were studied along with 5 nonatopic patients. All tests were conducted in a double-blind fashion with 6 determinations for each of 10 food antigens. Reproducibility of the test ($\text{5}\text{6}$ positive or negative) was demonstrated with wheat, milk, yeast, chocolate, and orange. In the nonatopic group, reproducible results were obtained for wheat, egg, yeast, chocolate, and chicken. Clinical correlation with 11 foods in 7 patients was demonstrated. However, there were 46 positive tests without correlation and 2 negative tests with positive histories. Therefore, there appears to be reproducibility of the tests to only 3 foods but no apparent correlation with clinical symptoms. At the present time, cytotoxic tests offer no reliable help in establishing the diagnosis of food allergy.     

Infection of chick cells by subgroup E viruses.

Studies were carried out to determine whether there is a restriction of replication of the endogenous chicken leukosis virus Rous associated virus type O (RAV-0) in chick embryo fibroblasts (CEF) beyond that imposed by the known cell surface barrier. Following either Sendai virus mediated fusion of RAV-O with surface resistant CEF ($\text{C}\text{E}$ cells), or infection of CEF lacking the surface barrier ($\text{C}\text{O}$ cells), a quantitative 10³- to 10⁴-fold restriction in replication was noted in comparison with RAV-60, a recombinant leukosis virus bearing the same subgroup E envelope as RAV-0 The failure of this internal restriction to operate against subgroup E leukosis viruses was investigated in detail in a series of cloned subgroup E sarcoma virus recombinants isolated following mixed infection with RAV-0 and Prague strain of Rous sarcoma virus subgroup C (PR-C) (i.e., two factor crosses) or following PR-C infection of chf⁺ CEF. RNA from one of these PR-E clones was shown to contain a nearly full complement of RAV-0 specific and RSV specific nucleotide sequences, but that isolate and six others were all free of the restriction observed with RAV-0 replication. One clone may be subject to some restriction. Thus some genetic function(s) of RAV-0 lost or inoperative in recombinant viruses appears important for this restriction. Since RAV-0 can replicate to a “normal” titer in some CEF from particular lines of chickens (line 7, line 100 × 7, and line 15), but apparently not in embryo culture from chicken flocks used in these studies, some cell function(s) also appears important for this restriction.     

Feeding pattern and light-dark variations in water intake and renal excretion after suprachiasmatic nuclei lesions in rats

Bilateral destruction of the suprachiasmatic nuclei (SCN) eliminated light-dark (L/D) variations in water intake and urine output in albino rats. The lesions abolished also the circadian rhythm of food intake, without changing significantly the 24 hour number of meals, total meal duration and 24 hour food intake. Only the L/D distribution of the number of meals was changed from $\text{5.6}\text{16.9}$ in control period to $\text{12.7}\text{12.9}$ after lesions. In contrast, the L/D distribution of sodium, potassium and chlorides excretions demonstrated attenuated but persistent nocturnal type. These data imply that SCN play a role of driving oscillator for the circadian rhythm of food intake, but probably are not the main synchronizer for the rhythms of electrolyte excretions.     

HLA-D related (DR) antigens in various kinds of myositis

Forty-eight patients with various kinds of myositis were studied for HLA-A,B.C. and DR antigens and compared to both local and 7th International Workshop controls. No A.B. or C antigens were increased except for B8 in white patients ($\text{6}\text{9\%}\text{or}\text{67\%)}\text{p}\text{= 0.005,}\text{pc}\text{=}\text{NS}$)_ and for B7 in black patients ($\text{6}\text{9}\text{or}\text{67\%) (}\text{p}\text{= 0.02,}\text{pc}\text{=}\text{NS}$). HLA-DR3 occurred in $\text{8}\text{12}$. or 67%, of whites with polymyositis (PM) as compared to 23% of white controls (p=0.003, pc=0.02). HLA-DRω6 was found in $\text{7}\text{9}$, or 78%, of blacks with PM compared to 26% of black local controls (p=0.005, pc=0.04) and 38% of black Workshop controls (p=0.005, pc=NS). Dermatomyosis (DM) patients, however, showed no increased frequencies of any HLA antigens. HLA-DR4 was decreased overall (p=0.005, pc=0.04). The immunogenetic differences between PM and DM suggest that they are pathogenetically different disorders.     

Ischemia-induced canine myocardial lysosome labilization: The role of endogenous prostaglandins and cyclic nucleotides

We tested the hypothesis that alterations in myocardial prostaglandin levels were associated with ischemia-induced lysosome labilization. To test this hypothesis, endogenous prostaglandin synthesis was restricted by administering indomethacin (INDO). After a thoracotomy in dogs, INDO (5 mg/kg) or vehicle was infused iv and 15 min later myocardial ischemia was induced by occlusion of the left anterior descending (LAD) coronary artery and the results compared to sham-operated animals. Myocardial tissue samples were biopsied 3 hr post-LAD ligation, homogenized, and the lysosome fraction subjected to a mild hypo-osmotic shock. The lysosomes from ischemic tissue were more labile compared to lysosomes from nonischemic tissue and this was associated with a decrease in myocardial $\text{cAMP}\text{cGMP}$ in ischemic tissue. Myocardial $\text{cAMP}\text{cGMP}$ correlated positively with myocardial lysosome stability (P < 0.05) and tissue prostaglandin E and A concentrations correlated negatively with myocardial cAMP (P < 0.05). In an in vitro liver lysosome system, INDO had no effect on enzyme release. These data suggest that ischemia-induced lysosome labilization via an increased prostaglandin release causes a lowered $\text{cAMP}\text{cGMP}$.     

Reversible inhibition of spermatogenesis in rats and monkeys with a new class of indazol-carboxylic acids

The effect of oral administration of $\text{AF 1312}\text{TS}$ upon the testicular germinal epithelium was studied in the rat and monkey. A single oral dose of 100 or 200 mgm/kgm given to mature male rats was not effective, but five consecutive doses of 200 mgm/kgm produced marked decrease in testicular weight and complete inhibition of spermatogenesis, while the weight and histology of the prostate and seminal vesicles were not affected. Daily doses of 10 mgm/kgm for 37 weeks or five consecutive doses of 50 mgm/kgm for 1 week were ineffective in the monkey. However, when the five dose regimen was followed by single weekly doses of 50 mgm/kgm for 6 months, complete inhibition was achieved and maintained in the monkey after 8 weeks. Daily doses of 100 mgm/kgm for 6 months resulted in inhibition of spermatogenesis.Preliminary studies with AF 1890 (an analog of $\text{AF 1312}\text{TS}$) given at levels of 50 mgm/kgm for 5 days to rats resulted in complete inhibition of spermatogenesis. The activity of this analog was four times greater than $\text{AF 1312}\text{TS}$. In monkeys, a daily dose of 200 mgm/kgm for 2 weeks also resulted in suppression of spermatogenesis.