Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

The biogeochemistry of potassium at Hubbard Brook

A synthesis of the biogeochemistry of K was conducted during 1963–1992 in the reference and human-manipulated watershed-ecosystems of the Hubbard Brook Experimental Forest (HBEF), NH. Results showed that during the first two years of the study (1963–65), which coincided with a drought period, the reference watershed was a net sink for atmospheric inputs of K. During the remaining years, this watershed has been a net source of K for downstream ecosystems. There have been long-term declines in volume-weighted concentration and flux of K at the HBEF; however, this pattern appears to be controlled by the relatively large inputs during the initial drought years. Net ecosystem loss (atmospheric deposition minus stream outflow) showed an increasing trend of net loss, peaking during the mid-1970s and declining thereafter. This pattern of net K loss coincides with trends in the drainage efflux of SO₄ ^2– and NO₃ ^–, indicating that concentrations of strong acid anions may be important controls of dissolved K loss from the site. There were no long-term trends in streamwater concentration or flux of K. A distinct pattern in pools and fluxes of K was evident based on biotic controls in the upper ecosystem strata (canopy, boles, forest floor) and abiotic controls in lower strata of the ecosystem (mineral soil, glacial till). This biological control was manifested through higher concentrations and fluxes of K in vegetation, aboveground litter, throughfall and forest floor pools and soil water in the northern hardwood vegetation within the lower reaches of the watershedecosystem, when compared with patterns in the high-elevation spruce-fir zone. Abiotic control mechanisms were evident through longitudinal variations in soil cation exchange capacity (related to soil organic matter) and soil/till depth, and temporal and disturbance-related variations in inputs of strong-acid anions. Marked differences in the K cycle were evident at the HBEF for the periods 1964–69 and 1987–92. These changes included decreases in biomass storage, net mineralization and throughfall fluxes and increased resorption in the latter period. These patterns seem to reflect an ecosystem response to decreasing rates of biomass accretion during the study. Clearcutting disturbance resulted in large losses of K in stream water and from the removal of harvest products. Stream losses occur from release from slash, decomposition of soil organic matter and displacement from cation exchange sites. Elevated concentrations of K persist in stream water for many years after clearcutting. Of the major elements, K shows the slowest recovery from clearcutting disturbance.     

The conserved 7SK snRNA gene localizes to human chromosome 6 by homolog exclusion probing of somatic cell hybrid RNA.

Many small RNAs contribute essential activities to eukaryotic cells. In mammalian genomes dispersed repetitive sequences which exhibit homology to small RNAs often exist as pseudogenes which can complicate identification, localization, and analysis of the authentic gene. We mapped a productive human 7SK small nuclear RNA gene to human chromosome 6 by analyzing Northern blots derived from a panel of somatic cell hybrids that contain single human chromosomes. In order to avoid crossreactivity of the probe with rodent 7SK RNA, which is 98% identical to human 7SK, a method termed homolog exclusion probing was developed. This method uses an excess of non-labelled rodent-specific oligodeoxynucleotide to block the rodent 7SK RNA from hybridizing with the human-specific oligodeoxynucleotide probe. The effectiveness of this method to enhance the human 7SK RNA signal is demonstrated. The potential to map and subsequently isolate other small RNA genes by this approach and the use of homolog exclusion probing to discriminate among family members of highly related RNAs and DNAs in a single species is discussed.     

DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.     

Feasibility of Outpatient Self-Administration of Parenteral Antibiotics

It is customary to treat patients with infective endocarditis or osteomyelitis for a prolonged period in hospital with parenteral antibiotics. It was felt that it might be feasible to allow parenteral administration of antibiotics by the patients themselves at home. Results in 13 patients who administered antibiotics parenterally themselves (experimental group) were compared with those in 7 patients (control group) treated entirely within the hospital. Antibiotic-related complications were similar in both groups. There was no instance of infection of the intravenous cannula in either group. The average daily cost of antibiotic therapy decreased from $243.22 for inpatients to $69.35 for outpatients. The average cost of illness was $6,357.22 in the experimental group and $10,022.23 in the control group. If patients are carefully selected and well educated, the outpatient self-administration of antibiotics parenterally is both economical and safe.     

Non-Hodgkin's lymphomas in Saskatchewan: a clinicopathologic study

In a retrospective clinical study of 208 previously untreated persons with non-Hodgkin''s lymphomas the disorders were classified and staged according to the histopathologic criteria of Rappaport, Winter and Hicks and the Ann Arbor clinical staging classification.Nodular types constituted 22% and diffuse types 78% of the lymphomas. The nodular lymphomas were slightly more common in females and were clustered in the age range 30 to 90 years. The diffuse lymphomas were slightly more common in males; the age distribution was bimodal, with one peak in the age range 10 to 19 years and the other in the age range 60 to 69 years, but when the age distribution of the general population in which the lymphomas occurred was taken into account, the incidence of these lymphomas was found to be significantly higher (P < 0.001) in persons more than 69 years of age than in those 40 to 69 years of age.Survival correlated with histopathologic type: persons with nodular (follicular) lymphomas and diffuse lymphocytic well differentiated lymphomas had a significantly greater survival (P < 0.05) than those with other diffuse lymphomas. No significant difference in survival was noticed between persons with nodal and extranodal lymphomas.While Rappaport and colleagues'' criteria are still very useful, it is important to recognize the nodular lymphoma as a specific entity requiring generally different management from diffuse lymphomas. Appreciation of the different biologic behaviour of the various lymphomas is important to clinicians planning therapy.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans.

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death, which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insight into the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells.