We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Chimaeric ribonuclease genes that are expressed in the anthers of transformed tobacco and oilseed rape plants were constructed. Chimaeric ribonu clease gene expression within the anther selec tively destroys the tapetal cell layer that surrounds the pollen sac, prevents pollen formation, and leads to male sterility. These nuclear male sterility genes should facilitate the production of hybrid seed in various crop plants.

We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric p-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional leve1 and that a 122-base pair 5' region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Injury severity determines the degree of IL-1beta protein level elevation after TBI. The effects of posttraumatic hypothermia on IL-1beta protein levels (an important mediator of neurodegeneration after TBI) may partly explain the established effects of posttraumatic temperature manipulations on inflammatory processes after TBI.

Recent evidence suggests that matrix metalloproteinases (MMPs) contribute to acute edema and lesion formation following ischemic and traumatic brain injuries (TBI). Experimental and clinical studies have also reported the beneficial effects of posttraumatic hypothermia on histopathological and behavioral outcome. The purpose of this study was to determine whether therapeutic hypothermia would affect the activity of MMPs after TBI. Male Sprague-Dawley rats were traumatized by moderate parasagittal fluid-percussion (F-P) brain injury. Seven groups (n=5/group) of animals were investigated: sham-operated, TBI with normothermia (37 degrees C), and TBI with hypothermia (33 degrees C). Normothermia animals were killed at 4, 24, 72 h and 5 days, and hypothermia animals at 24 or 72 h. Brain temperature was reduced to target temperature 30 mins after trauma and maintained for 4 h. Ipsilateral and contralateral cortical, hippocampal, and thalamic regions were analyzed by gelatin and in situ zymography. In traumatized normothermic animals, TBI significantly (P<0.005) increased MMP-9 levels in ipsilateral (right) cortical and hippocampal regions, compared with contralateral or sham animals, beginning at 4 h and persisting to 5 days. At 1, 3, and 5 days after TBI, significant increases in MMP-2 levels were observed. In contrast to these findings observed with normothermia, posttraumatic hypothermia significantly reduced MMP-9 levels. Hypothermic treatment, however, did not affect the delayed activation of MMP-2. Clarifying the mechanisms underlying the beneficial effects of posttraumatic hypothermia is an active area of research. Posttraumatic hypothermia may attenuate the deleterious consequences of brain trauma by reducing MMP activation acutely.

Rapid and marked increase in TNFalpha mRNA expression and protein levels follows moderate and severe TBI. Injury severity and posttraumatic temperature play a modest but significant role on TNFalpha expression and protein levels. These findings suggest that the effects of posttraumatic temperature on histopathological and behavioral outcome primarily may involve secondary mediators that do not operate directly through their effect on TNFalpha.

Therapeutic hypothermia promotes protection after traumatic brain injury (TBI). The mechanisms underlying hypothermic protection are multifactorial and may include the modulation of microRNA (miRNA) expression after trauma. We utilized microarrays to examine the effects of posttraumatic hypothermia on the expression of 388 rat miRNAs. Animals were subjected to sham or moderate fluid percussion brain injury, followed by 4 hours of hypothermia (33°C) or normothermia (37°C) and euthanized at 7 or 24 hours. At 7 hours, 47 miRNAs were significantly different (P<0.05) between TBI and sham (15 higher in TBI and 31 lower). After 24 hours, 15 miRNAs differed by P<0.05 (7 higher and 9 lower). The expression of miRNAs was altered by posttraumatic hypothermia. At 7 hours, seven were higher in hypothermia than normothermia and five were lower. Some miRNAs (e.g., miR-874 and miR-451) showed the most difference with hypothermia, with changes verified by quantitative reverse transcriptase-PCR. Regionally specific miRNAs also showed responses to TBI and hypothermia treatments by in situ hybridization. In addition, in vitro neuronal stretch injury studies showed similar temperature-sensitive responses to specific miRNAs. These novel data indicate that the reported beneficial effects of early hypothermia on traumatic outcome may include temperature-sensitive miRNAs involved in basic cell-processing events.

The proinflammatory cytokine interleukin-1β (IL-1β) is induced rapidly after traumatic brain injury (TBI) and contributes to the inflammatory events that lead to neuronal loss. Although an important source of IL-1β is from the injured brain itself, in patients with multiple organ trauma (polytrauma) IL-1β is also released into the bloodstream which may potentially influence brain vulnerability. The purpose of this study was to determine the effects of systemic inflammation induced by peripheral administration of IL-1β on histopathological and behavioral outcome after moderate fluid percussion (FP) brain injury in rats. At 30 min or 24 hr after TBI, saline, 20μg/kg or 40μg/kg of IL-1β was injected (n=4-9/group) intraperitoneally (IP). Sham operated animals (n=9) received either saline or IL-1β (20 or 40 μg/kg) injections. The somatosensory tactile placing test was administered at 1, 2 and 3 days posttrauma. IL-1β treated animals showed significant placing deficits compared to vehicle-treated TBI animals. Three days after injection, contusion areas and volumes were significantly increased (p<0.05) with both IL-1β doses and at both treatment times compared to vehicle treated animals. IL-1β treated rats showed more contusion injury and hippocampal neuronal damage as well as enhanced perivascular neutrophil accumulation. Cortical IL-1r1 mRNA increased as early as 1 hr following TBI, peaking at 24 hr and remained elevated 3 days posttrauma. These data show that the posttraumatic administration of IL-1β significantly aggravates behavioral outcome and increases overall contusion volume after TBI. Increased systemic inflammatory processes, including extravasation of activated leukocytes and proinflammatory cytokines could participate in this detrimental outcome. Because peripherally circulating cytokines and other neurotoxic factors may be increased following multi-organ trauma, these findings may be important in targeting therapeutic interventions in this patient population.