Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP₂, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP₃, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.     

Virus-enhanced modulation of cell surface antigens: effect on immune lytic susceptibility.

Monkey kidney cells, upon progressive subculture, became refractory to complement (C)-dependent immune cytolysis by anti-cell serum. Arbovirus infection restored these cells to a state of lytic susceptibility. Similar results were also abtained with antibody-dependent cellular cytotoxicity (ADCC), which is C independent. Antibodies raised against different subcultures varied considerably in lytic efficiency, indicating changing patterns of host cell expression during continous subculture. Taken together with the fact that arbovirus infection festored the lytic efficiency of all antibody preparations to the same degree suggested some form of host cell antigen re-expression as a mechanism. The results obtained in several exploratory experiments indicated that the antigenic re-expression responsible for the restoration of lysis was probably a local or selective rather than a generalized phenomenon. Thus, the amount of host cell surface antigen, measured by the use of mouse anti-cell serum and 125I anti-mouse globulin, was identical in both uninfected lytic susceptible and refractory cells, and decreased in both functional states following infection. Further, the binding of 125I concanavalin A, used to quantify surface glycoproteins, was similar in both lytic refractory and susceptible cells, and in both cases declined folowing virus infection. This result was incompatible with gross "masking" of cell surface antigens by exuberant production of surface coat material in lytic resistant cells. Finally, brief trypsinization of lytic resistant cells yielded an 8-fold increase in immune lysis, a result further consistent with local rather than generalized surface changes. The data were discussed interms of modulation of cell surface antigens affected both by repeated subculture and arboviral infection, and as a possible in vitro correlate of altered self-reactivity.     

TRANSMITTER METABOLIZING ENZYMES AND FREE AMINO ACID LEVELS IN SENSORY AND MOTOR NERVES AND GANGLIA OF THE SHORE CRAB (CARCINUS MAENAS)

—The distribution of ChAT (choline acetyltransferase), GAD (glutamate decarboxylase) and acetylcholinesterase in some sensory and motor nerves of the shore crab, Carcinus maenas, has been investigated using micro-assay techniques. ChAT was concentrated in the afferent nerve fibres of the thoracic-coxal muscle receptor as well as in the coxo-basal chordotonal receptor nerve and other leg sensory fibres. GAD was found in leg motor nerves including the promotor and remotor muscle nerves, being undetectable in the sensory nerves. Acetylcholinesterase was found in similar levels in both sensory and motor nerves assayed. Amino acid analysis using a micro-dansylation technique showed that sensory nerves had low GABA levels, whereas the leg nerve including motor fibres had substantially higher GABA concentrations. GAD and GABA were also found in low amounts in the leg promoter mucle, which is consistent with GABA being a neuromuscular transmitter.     

Frequency response characteristics of electroreceptors in weakly electric fish (Gymnotoidei) with a pulse discharge

Summary Three species of Gymnotid fish, two species ofHypopomus andRhamphichthys rostratus, each having pulse type electric organ discharges (EOD) of different durations were studied to learn if any correlation exists between the spectral composition of the species specific EOD pulse and the frequency response characteristics of that species' electroreceptors. The receptor population consisted of two major categories (examples in Fig. 3). One category, termed pulse marker receptors, responded to suprathreshold stimulus pulses with a single spike at a short (<2 ms) latency. These receptors were tuned to the higher frequency components of a species' EOD (Fig. 4A) and were always 5 to 10 dB less sensitive than any other electroreceptors within a given species. The second major receptor category, burst duration coders, responded to an electrical stimulus with a burst of spikes at a longer latency, burst length was a function of stimulus amplitude. This second category could be further divided into three sub-categories according to the receptors' frequency response characteristics. The most commonly seen subcategory, wide band receptors (Fig. 4B), responded best to stimuli having frequencies equal to the dominant frequency component of the species' EOD in the two species ofHypopomus studied. A second subcategory, narrow band receptors (Fig. 4 A), had frequency response characteristics similar to those of the pulse marker receptors; however, these had thresholds 10 dB lower than those of the pulse marker. The third subcategory of burst duration coders, low frequency receptors (Fig. 4 C, D), responded best to stimulus frequencies ranging from about 50 to 150 Hz. Mechanisms of coding stimulus amplitude and responses to prolonged sinusoidal electrical stimuli were also studied in the various receptor types.It is suggested that the differences in the major receptor types and the different frequency response characteristics of the electroreceptors within a given species allows the animals to identify and evaluate signals resulting from their own EOD, the EODs of conspecifics and electrical stimuli generated by other species of electric fish.Supported by NIH Grant #1 RO1 NS 12337-01     

Differential replication of satellite DNA in polyploid tissues of Drosophila virilis

Satellite DNA amounts were examined in adult tissues of Drosophila virilis, a species whose DNA contains three prominent satellites. Satellite amounts in DNA from six of the seven tissues were lower than in DNA from diploid (adult brain) tissue. Satellite amounts in adult ovary DNA, however, were equivalent to or greater than diploid levels. When DNA from pupal ovaries was examined, a 30% increase in satellite amounts over diploid levels was found. An RNA-DNA hybridization experiment showed that the ribosomal RNA genes in pupal ovary DNA were under-replicated relative to diploid DNA levels.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.     

Studies on the Survival and Fate of Enteroviruses in an Experimental Model of a Municipal Solid Waste Landfill and Leachate

In laboratory scale municipal solid waste lysimeters containing simulated refuse, and seeded with either laboratory or field strains of poliovirus type 1 and echovirus type 7, viruses were not detected in the lysimeter leachate produced over a 4-month period. In addition, viruses were detected in the lysimeter refuse contents after termination of lysimeter operation. These results appeared to be due to virus retention in the lysimeter caused by virus adsorption and virus inactivation. Evidence for virus inactivation was provided by the results of experiments on virus inactivation in composite leachate samples. Evidence for virus adsorption was supported by the rapid adsorption of viruses to various municipal solid waste components in the presence of a salt similar in composition to the major inorganic salts of leachates.