Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.     

Studies of bovine genital tracts: Mycoplasma isolations from dairy cows

Mycoplasma was isolated from the genital tracts of 8 of 96 culled dairy cows. Procedures were used to reduce the possibility of uterine contamination by vaginal fluids. The mycoplasma isolates were identified by the fluorescent antibody technique as: $\text{Mycoplasma bovigenitalium}$ (4), $\text{M. canadense}$ (1), an untyped bovine pathogen from the mammary gland designed as ST-6 (2), and an isolate that could not be identified with the labeled antibodies used in this study. All isolations were made from vaginal swabs, except in one case of pyometra where $\text{M. bovigenitalium}$ was isolated concurrently from the uterus and vagina, along with $\text{Pasteurella hemolyticum}$. These results suggest that mycoplasma may be found in the cervico-vaginal flora of some cattle but are rarely isolated from the uterine environment.     

Effect of Brucellaabortus infection on spermatogenesis in three Zebu bulls (Bosindicus). A case report

This case report addresses the occurrence of Brucellosis and its effect on the cattle in developing countries. Three Zebu bulls ($\text{Bos}$$\text{indicus}$) are presented and the clinical and pathologic signs are described. Conception rates declined following an abortion storm in one herd and without prior abortions in another herd. Semen collected by electro-ejaculation was found to be azoospermic or with very few spermatozoa. $\text{B. abortus}$ was isolated from seminal vesicles, testes and epididymides. Organs affected and showing microscopic lesions were testes, epididymides and seminal vesicles. The latter were not consistently affected. None of the bulls showed impairment of libido or breeding capacity.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

Alkyl glucosides as effective solubilizing agents for bovine rhodopsin. A comparison with several commonly used detergents

The suitability of octyl and decyl-β-d-glucoside as solubilizing agents for the bovine retinal rod outer segment disc membrane was investigated and compared to that of hexadecyltrimethylammonium bromide, $\text{N,N-}\text{dimethyldodecylamine}$ oxide, Emulphogene BC-720 and digitonin. The properties measured included the thermal stability of rhodopsin, regenerability of bleached rhodopsin by addition of $\text{11-cis-}\text{retinal}$, and the rate of denaturation of bleached rhodopsin as measured by changes in the ultraviolet CD spectrum. Denaturing tendencies of the detergents were also evaluated by observing their effects on the absorption and CD spectra of sperm whale metmyoglobin. Our results demonstrate that octyl glucoside is superior to the other detergents, with the possible exception of digitonin, by the above criteria. Unlike digitonin, however, octyl glucoside affords rapid solubilization of the disc membrane and is itself highly soluble. Decyl glucoside has properties equivalent or superior to octyl glucoside, but salts and buffers interfere with its ability to solubilize the disc membrane. The well defined chemical composition, ease of removal by dialysis, and non-denaturing properties of the alkyl glucosides make them attractive detergents for membrane research.     

Babesia major in Britain: blood-induced infections in splenectomized and intact calves

A strain of Babesia major, originally isolated from field collections of Haemaphysalis punctata in Kent, England was maintained in splenectomized calves by the intravenous inoculation of infected blood. Rapid passage from carrier calves, that had recovered from a tick-induced infection, resulted in a marked increase in virulence; 4 out of 6 calves of the second passage underwent fatal infections and the others suffered severe reactions.Five splenectomized and 5 intact calves of the same breed and age were infected with the same number of infected erythrocytes (RBC). The intact calves reacted mildly with maximum parasite counts ranging from < $\text{1}\text{1000}$ RBC to $\text{5}\text{1000}$ RBC; haemoglobin levels and packed cell volume values, however, fell sharply but recovered swiftly. The group of splenectomized calves exhibited one fatal case, 2 severe reactions and 2 mild infections; maximum parasitaemias varied from $\text{7}\text{1000}$ RBC to $\text{322}\text{1000}$ RBC. Packed cell volumes and haemoglobin concentrations declined to low levels and took several weeks to return to normal.It is concluded that B. major should be regarded as a potential pathogen of British cattle.     

Isolation, identification and stimulatory activity of a second component of the sex pheremone system (complex) of the female almond moth, Cadra cautella (Walker)

$\text{Cis}$-9-tetradecen-l-ol acetate was isolated from both extracts of female abdominal tips and of filter paper exposed to female $\text{Cadra}$$\text{cautella}$ (Walker). It is the second sex stimulant isolated from this species and, in stimulatory bioassays, it is ca. 1/100, 000 as active as the stimulant isolated previously, $\text{cis}$-9, $\text{trans}$-12-tetradecadien-l-ol acetate. According to attractancy tests, the natural sex pheromone system of complex contains other components in addition to these 2 stimulants.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

Sex pheromone of the potato tuberworm moth, Phthorimaea operculella.

A compound was isolated from female $\text{Phthorimaea operculella}$ (Lepidoptera: Gelechiidae) abdominal tip extracts and identified as $\text{trans}$-4, $\text{cis}$-7-tridecadienyl acetate. The synthetic compound elicits male $\text{P. operculella}$ wing fanning and upwind orientation in the laboratory, and is attractive to males in the field. The corresponding alcohol also appeared to be present in the female extracts, but this compound was not found to increase male responses in the laboratory or the field. Another EAG active component was isolated but not identified.     

Culture of bovine embryos after invitro exposure to Brucellaabortus

Fifty-four day-6 through day-10 (estrus=day 0) embryos were collected nonsurgically from 13 superovulated, brucellosis-free mixed breed cows. Forty-eight excellent and good zona pellucida-intact (ZP-I), three zona pellucida-defective (ZP-D), and three zona pellucida-free (ZP-F) embryos were incubated in media containing $\text{Brucella}$$\text{abortus}$. Subsequently, embryos were washed ten times in groups of one, two, three, or four. Embryos and serial washes were cultured for $\text{B}$. $\text{abortus}$.Brucellae were not isolated from any ZP-I embryo or from any washing beyond the sixth serial wash. Brucellae were not isolated from the three ZP-F embryos but were detected in the eighth wash for one and in the tenth wash for the others. Brucellae were isolated from one of three ZP-D embryos. Results show that ZP-I embryos can be effectively washed free of $\text{B}$. $\text{abortus}$.     

The effect of visible light on the regeneration of rhodopsin

Both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$, increased exposure to visible light decreases the regenerability of the visual pigment. Isolated opsin irradiated with increasing periods of white light decreased in pigment formation yields on combination with 9- or 11-$\text{cis}$ retinal. The yield of regeneration of the visual pigment extracted from albino rats depended on the amount of light to which the animal had been exposed. Animals exposed to normal room light demonstrated lower regeneration yields than dark-reared animals, but these yields increased on dark adaption. Opsin from animals exposed to sunlamps did not regenerate any pigment. On dark adaption, the pigment yields increased but the opsin level remained below that for the control group.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Evidence for plastoquinol-cytochrome f/b-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria

Membranes isolated from $\text{Nostoc}$ sp. strain MAC and $\text{Anacystis}\text{nidulans}$ displayed spectral changes in the cytochrome $\text{f}\text{b}$ region when examined by reduced $\text{minus}$ oxidized or dual wavelength spectrophotometry under physiological conditions. The same changes accompanied both light-induced (photosynthetic) and oxygen-induced (respiratory) electron transport. Physiological reduction of the cytochrome $\text{f}\text{b}$ moiety was abolished after extraction of plastoquinone but reappeared on reconstitution of the depleted membranes with authentic plastoquinone. Moreover, a mutual inhibition of photosynthetic and respiratory activities could be directly demonstrated with the isolated membranes. From the results it is concluded that the membrane-bound plastoquinol-cytochrome $\text{f}\text{b}$ reductase functions as a common electron donor to both P700 and the cytochrome oxidase in cyanobacteria.     

Calcium content of frog rod outer segments and discs

To test the “Ca²⁺ hypothesis of visual excitation”, we measured the total Ca²⁺ content of freshly isolated bullforg rod outer segments, and have compared the total Ca²⁺ contents of fully dark-adapted discs with discs exposed to small amounts of light. Discs were prepared by hypotonically lysing outer segments under conditions expected to remove Ca²⁺ from the cytoplasm but not from the discs. Ca²⁺ was assayed by atomic absorption spectrophotometry. We find that both discs and outer segments contain a total of about 0.1–0.2 Ca²⁺ per rhodopsin molecule. Thus, each frog disc retains about $\text{2 · 10}{}^5\text{Ca}{}^{\mathrm{2+}}$. If most of this Ca²⁺ were free in the aqueous space inside the intact discs, the Ca²⁺ activity would be a few mM. Since the light-regulated Na⁺ channels have been reported to be highly sensitive to cytoplasmic Ca²⁺, this store of Ca²⁺ in the discs is far more than required by the Ca²⁺ hypothesis. However, despite several variations in experimental conditions, we did not observe any light-activated release of Ca²⁺ from discs in response to stimuli that photoactivated a small fraction of the rhodopsin, as required by the Ca²⁺ hypothesis. In the 26 experiments reported here we could have detected a release as small as 20–30% of the Ca²⁺ content of the disc.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.     

Effects of 6-thioguanine-containing RNA on in vitro protein synthesis

6-Thioguanine was administered to rats 12 hr after partial hepatectomy at a dose of 40 mg/kg of body weight; 6 hr later, polyadenylic acid-containing RNA was isolated and was used to measure initiation of protein synthesis $\text{in vitro}$ in a wheat germ system. $\text{In vitro}$ initiation was found to be 2.3-fold greater when 6-thioguanine-containing RNA was employed, than when polyadenylic acid-containing RNA isolated from untreated animals was used. The homopolymer, poly(TG), did not promote peptide synthesis in the wheat germ $\text{in vitro}$ system employed.     

Plasma progesterone concentration in Bos taurus and Bos indicus heifers

Progesterone concentrations were determined by radioimmunoassay in the plasma of heifers of German Brown Swiss, Holstein-Friesian and White Fulani cattle during two consecutive estrous cycles in the dry and wet seasons in a tropical climate. Seasonal differences in the levels were not significant but peak levels were significantly higher in the $\text{Bos}$$\text{taurus}$ than in the $\text{Bos}$$\text{indicus}$ heifers. The low levels at estrus and the gradual rise five days after estrus were similar among the heifers. The levels on the 1st and 2nd days preceding estrus were significantly higher in the $\text{Bos}$$\text{taurus}$ than in the $\text{Bos}$$\text{indicus}$ heifers, and suggest a possible tendency for the former to be more susceptible to infertility arising from high progesterone levels 1 and 2 days preceding estrus and insemination. The genital tract and ovaries at the mature luteal phase were generally smaller in the $\text{Bos}$$\text{indicus}$ than in the $\text{Bos}$$\text{taurus}$ heifers.     

Characterization of DNA from Rhizobium cells and their bacteriods from root nodules

DNA from two rhizobial strains $\text{Cicer}$ and $\text{Phaseolus}$ and their bacteroids from root nodules have been isolated, purified and characterized for thermal denaturation temperature and buoyant density. Bacteroid DNA had a lower Tm value and buoyant density comparad to $\text{Rhizobium}$ cell DNA. The calculated GC content of becteroid DNA was lower than the $\text{Rhizobium}$ cell DNA.